Osteoclast differentiation inhibitors

ABSTRACT

A compound represented by formula (I); a process for producing a compound (substance F-1490) of formula (I), wherein X represents —O— and R represents a hydroxyl group, by using a microorganism belonging to the genus  Cunninghamella;  and a  Cunninghamella  sp. F-1490 strain (FERM BP-8287) capable of producing the substance F-1490; and an osteoclast differentiation inhibitory agent containing as an active ingredient the compound represented by formula (I):  
                 
 
wherein X represents —O— or —CH 2 —, and R represents a hydroxyl group when X represents —O—, or a hydrogen atom when X represents —CH 2 —.

TECHNICAL FIELD

The present invention relates to a compound represented by formula (I):

wherein the symbols have the same meanings as described hereinbelow, aprocess for producing a compound (substance F-1490) of formula (I),wherein X represents —O— and R represents a hydroxyl group, by using amicroorganism belonging to the genus Cunninghamella; a Cunninghamellasp. F-1490 strain (FERM BP-8287) capable of producing the substanceF-1490; and an osteoclast differentiation inhibitory agent comprisingthe compound represented by formula (I) as an active ingredient.

BACKGROUND ART

Osteoclasts are cells generated by differentiation of bone marrow cellsand known to be involved in a number of diseases such as osteometastasisof cancer cells including breast cancer cells, rheumatoid arthritis, andosteoporosis. Accordingly, it is presumed that successful suppression ofthe differentiation from bone marrow cells into osteoclasts will resultin excellent therapeutic effects on the aforementioned diseases. Thus,it has been desired to develop substances that have low toxicities andexhibit potent inhibitory effects against the differentiation from bonemarrow cells into osteoclasts. However, no reports have been issueduntil now on substances having such properties as mentioned above amonglow molecular weight substances derived from microorganisms.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a novel compoundthat has low toxicities and exhibits an osteoclast differentiationinhibiting effect; a process for producing the compound; a novelmicroorganism having the capability of producing such a compound; and anosteoclast differentiation inhibitory agent containing as an activeingredient the compound.

Note that the term “osteoclast differentiation inhibition” as usedherein means inhibiting the differentiation from bone marrow cells intoosteoclasts.

To attain the above-mentioned object, the present inventors isolatedmicroorganisms from soils of various geographical areas and extensivelystudied metabolites produced by the microorganisms. As a result, theyhave found that a microorganism belonging to the genus Cunninghamellanewly isolated produced in the culture broth a substance that showsosteoclast differentiation inhibiting activity. Separation andpurification of the active substance from the culture broth andinvestigation of the physicochemical properties allowed the inventors toconfirm that the obtained active substance is a novel compoundrepresented by formula (I) described below that differs from any knownsubstance and has excellent osteoclast differentiation inhibitingactivity.

Further, the present inventors have synthesized novel analogue compoundsor formula (II) that have a structure similar to that of the compoundrepresented by formula (I) and examined the properties thereof. Theresults obtained allowed the inventors to confirm that the analoguecompounds had likewise osteoclast differentiation inhibiting activity atlow concentrations. The present invention has been achieved based onthese findings.

That is, the present invention relates to a novel compound havingosteoclast differentiation inhibiting activity described below or saltsthereof; a process for producing the compound; a microorganism thatproduces the compound; and a drug that contains as an active ingredientthe compound.

-   -   1. A compound represented by formula (I):        wherein X represents —O— or —CH₂—, and R represents a hydroxyl        group when X represents —O—, or a hydrogen atom when X        represents —CH₂—, or salts thereof.    -   2. The compound according to 1 above, wherein in formula (I), X        represents —O— and R represents a hydroxyl group, or salts        thereof.    -   3. The compound according to 1 above, wherein in formula (I), X        represents —CH₂— and R represents a hydrogen atom, or salts        thereof.    -   4. A process for producing the compound according to 2 above,        including cultivating a microorganism that belongs to the genus        Cunninghamella and is capable of producing the compound        according to 2 above, and recovering the compound according to 2        above from the resultant culture broth.    -   5. Cunninghamella sp. F-1490 strain having a capability of        producing the compound according to 2 above.    -   6. An osteoclast differentiation inhibitory agent containing as        an active ingredient the compound or salts thereof according to        1 above.

Hereinafter, the present invention will be described in detail.

The present inventors named a compound among the compounds representedby formula (I), in which X represents —O— and R represents a hydroxylgroup, a substance F-1490.

The substance F-1490 is a novel substance that is clearly distinguishedfrom known compounds by the molecular formula, physicochemicalproperties and structural characteristics.

Salts of the substance F-1490 of the present invention include saltswith pharmaceutically acceptable bases (inorganic bases and organicbases), for example, salts with inorganic bases such as sodium salt,potassium salt, ammonium salt, calcium salt, magnesium salt and aluminumsalt, salts with basic amino acids (for example, arginine, lysine, etc.)and so on.

Further, the present invention provides a process for producing thesubstance F-1490 by cultivating a microorganism belonging to the genusCunninghamella and having the capability of producing the substanceF-1490 that exhibits osteoclast differentiation inhibiting activity andrecovering the substance F-1490 from the resulting culture broth; amicroorganism belonging to the genus Cunninghamella that has thecapability of producing the substance F-1490; and osteoclastdifferentiation inhibitory agents that contain as an active ingredientthe substance F-1490.

The microorganism used in the present invention may be any microorganismas long as it is a strain that belongs to the genus Cunninghamella andhas the capability of producing the substance F-1490 of the presentinvention. Search for such a microorganism can be performed, forexample, as follows. That is, extracts of various microorganism culturebroths are added to culture broth of bone marrow cells and the number oftartaric acid resistant acid phosphatase-positive cells, which is anindex of differentiation into osteoclasts, is measured. The targetmicroorganism that has the capability of producing the substance F-1490can be obtained by isolating and identifying an active substance fromculture broth of the microbe that shows a decrease in the number ofcells, that is, shows inhibition of the differentiation intoosteoclasts.

Examples of the microorganism found in this manner include a strainF-1490 that was isolated from soil by the present inventors and belongsto the genus Cunninghamella. However, the microorganism is not limitedto this strain but all the strains can be used in the present inventionso far as they belong to the genus Cunninghamella and have thecapability of producing the substance F-1490 of the present invention,including mutants thereof, for example, artificial mutants obtained bytreatments with mutagens such as ultraviolet rays, X rays, radiationsand chemicals as well as spontaneous mutants.

Hereinafter, mycological properties of the strain F-1490 will beexplained.

Inoculation of this strain on potato dextrose agar (hereinafter,referred to as “PDA”), malt extract agar (hereinafter, referred to as“MEA”), oatmeal agar (hereinafter, referred to as “OA”) and cultivationat 25° C. resulted in wool-like hyphae, the color of which was initiallywhite (A1) and subsequently turned cream (4A3) via yellowish white (4A2)on all the agar plates.

The growth rate is extremely high; the hyphae covered all over thesurface of a dish of 85 mm in diameter in all the plates under thecondition of cultivation for three days. The growth is very vigoroussuch that after one week, the lid of the dish is lifted. Further, in allthe plates, no production of soluble pigments is observed. Note that thedescriptions on color tone are made according to Methuen Handbook ofColour (Kornerup & Wanscher, 1978).

The morphological characteristics of the strain are as follows. Under anoptical microscope, only peculiar sporangiophores are observed but notsporocarps. Aerial hyphae have few septa and also have stolons.Formation of rhizoids is also confirmed. Sporangiophores are formedmonogenically from aerial hyphae such as stolons. The stipes are smoothand grow linearly. The stipes have vesicles at their apices below whichirregular or whorled branchings are observed. The branchings have smallvesicles on their apices. The vesicles are spheroidal to oval with awidth of 30 μm at the apices or a maximum of 20 μm in the branchings.The strain has neither sporangium nor merosporangium but has unilocularsporangiolum that arises on the surface of the vesicles. Thesporangiolum is globose to ellipsoidal and is of a size of 6 to 9 μm.The sporangiolum is brown and its surface is short-echinulate. No striaare observed.

From the mycological properties mentioned above, the present inventorsjudged that the strain belongs to the genus Cunninghamella, named itCunninghamella sp. F-1490 and deposited it on Oct. 2, 2001 at theindependent administrative corporation, National Institute of AdvancedIndustrial Science and Technology, International Patent OrganismDepositary at Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,Japan, as the accession number of FERM P-18548, and transferred from theoriginal deposit to international deposit based on Budapest Treaty onJan. 31, 2003, and deposited as the accession number of FERM BP-8287.

The substance F-1490 of the present invention can be produced byinoculating the above-mentioned strain in a nutrient source-containingmedium and cultivating it aerobically. The substance F-1490 producingmicroorganism is not limited to the above-mentioned strain but all thestrains that belong to the genus Cunninghamella and have the capabilityof producing the substance F-1490 may be used in the present invention.

The method of cultivating the above-mentioned microorganisms is inprinciple pursuant to the cultivation method for general microorganisms.Usually, it is preferable that the method is practiced under aerobicconditions, such as shaking culture by liquid culture or aerationagitation culture. The medium that is used for cultivation maybe anymedium that contains nutrient source available to the microorganismsbelonging to the genus Cunninghamella. Various synthetic andsemi-synthetic media as well as natural media are available. Thecomposition of medium is as follows. Carbon sources including glucose,sucrose, fructose, glycerol, dextrin, starch, molasses and so on may beused singly or in combination. Nitrogen sources including organicnitrogen sources such as pharmamedia, peptone, meat extract, soybeanpowder, casein, amino acids, yeast extract and urea may be used singlyor in combination. In addition, salts such as sodium chloride, potassiumchloride, calcium carbonate, magnesium sulfate, sodium phosphate,potassium phosphate and cobalt chloride, heavy metal salts, vitaminssuch as vitamin B and biotin may be added as necessary.

In case considerable foaming occurs during the cultivation, variousdefoaming agents may be added in the medium as appropriate. When thedefoaming agent is added, care must be taken to add the defoaming agentin a concentration that does not adversely affect production of thetarget substance. It is desirable that the pH of the medium is about 5to about 9, usually around neutrality. The cultivation temperature isrecommended to be kept at usually 10 to 40° C., preferably 20 to 27° C.The cultivation time is about 2 to about 14 days, usually 3 to 5 days.Needless to say, various cultivation conditions described above may bevaried depending on the kind and characteristics of the microorganismused, external conditions and so on as appropriate and optimalconditions can be selected. The substance F-1490 of the presentinvention which accumulates in the culture broth can be recovered byseparating the fungus body by using known ordinary solid-liquidseparation means such as filtration and centrifugation; and extractingthe target compound from the filtrate.

Separation and purification of the substance F-1490 can be performed byselecting and combining various known methods. For example, a solventextraction method using ethyl acetate, n-butanol or the like, and acolumn chromatographic method using a carrier, for example,polystyrene-based adsorbent resin such as Amberlite XAD (manufactured byRohm and Haas Co., Ltd.) and Diaion HP-20 (manufactured by MitsubishiChemical Corporation), silica gel, alumina or activated carbon may beused. The method of eluting the target substance from the carriers mayvary depending on the kind and properties of the carrier. For example,in the case of polystyrene-based adsorbent resin, hydroalcohol,hydroacetone and the like can be used as eluting solvents. Further, gelfiltration using Sephadex LH-20 (manufactured by Pharmacia AB), Bio GelP-2 (manufactured by Bio-Rad Laboratories) or the like, thin layerchromatography using silica gel, alumina or the like, preparative highperformance liquid chromatography (preparative HPLC) using a normalphase or reversed phase column and so on may be used. These methods maybe used singly or in combination as appropriate, or in repetitions asappropriate to effect separation and purification.

The substance F-1490 thus obtained has the following physicochemicalproperties.

(1) Form: white powder,

(2) Molecular formula: C₁₂H₁₃NO6

(Measurement of C₁₂H₁₄NO₆ by high resolution FAB mass spectrometryCalculated m/z: 268.0821 (M+H)⁺, Found m/z: 268.0861),

(3) Specific optical rotation:[α]_(D) ²² −57.5° (c 0.4, methanol)

(4) Melting point: 125 to 131° C. (dec.)

(5) Infrared absorption spectrum: The results obtained by measurementsby the KBr method are as shown in FIG. 1. The characteristic absorptionsare as follows.

IRν_(max) (KBr) cm⁻¹: 3380, 1680, 1620, 1500, 1440, 1375, 1260, 1090,1035.

(6) Ultraviolet absorption spectrum: The results obtained bymeasurements in methanol are as shown in FIG. 2. The characteristicabsorptions are as follows.

UVλ_(max) nm: 217, 245 (sh), 328.

(7) Solubility: Readily soluble in ethyl acetate, methanol, dimethylsulfoxide and water; hardly soluble in hexane.

(8) ¹H-nuclear magnetic resonance spectrum: The results obtained byperforming measurement by dissolving the substance in heavy pyridine andusing tetramethylsilane as internal standard are as shown in FIG. 3.Chemical shift, multiplicity and spin coupling constant of each signalare as follows.

δ 8.01 (1H, dd, J=1, 8 Hz), 6.76 (1H, t, J=8 Hz), 5.12 (1H, s), 4.64(1H, m), 4.40 (1H, ddd, J=2,5,11 Hz), 3.78 (1H, dd, J=11,11 Hz), 3.30(1H, ddd, J=2,5,13 Hz), 2.45 (1H, dd, J=11,13 Hz).

(9) ¹³C-nuclear magnetic resonance spectrum: The results obtained bymeasurements as dissolved in heavy pyridine and using tetramethylsilaneas internal standard are as shown in FIG. 4. Chemical shift andmultiplicity of each signal are as follows.

δ 171.7(s), 142.6(s), 135.6(s), 124.8(d), 121.1(d), 116.6(d), 114.1(s),92.3(s), 81.2(d), 72.0(t), 64.1(d), 45.9(t).

Further, the present inventors synthesized a novel analogue having achemical structure similar to that of the substance F-1490, representedby formula (I) in which X represents —CH₂— and R represents a hydrogenatom (hereinafter, referred to as “F-1490-A”) (see Example 2 describedlater) and tested it together with the substance F-1490 for theirphysiological activity.

The substances F-1490 and F-1490-A of the present invention haveexcellent osteoclast differentiation inhibiting activity and in additionlow toxicity to cells, so that it is expected that they can be used astherapeutic agents for diseases that accompany an increase in theactivity of osteoclasts, for example, osteometastasis of cancer cells,rheumatoid arthritis, osteoporosis and so on.

The osteoclast differentiation inhibiting activity of the substances ofthe present invention can be measured, for example, by the methoddescribed below.

Bone marrow cells were collected from a C57BL/6mouse (female, 6 weeksold) and suspended in RPMI1460 medium that contains 10% fetal calfserum, 1 mM pyruvic acid, 0.1 mM non-essential amino acids (manufacturedby Gibco), 500 μM 2-mercaptoethanol, 200 ng/ml parathyroid hormonerelated protein and 50 μg/ml L-ascorbic acid and 1 ml of the suspensionadjusted to a cell population of 1.5×10⁶ cells/well was inoculated on a24-well microplate. At the same time a test sample was added inappropriate amounts. On days 2 and 4 from the start of the cultivation,half the amount of the medium is exchanged with fresh medium containingthe test sample. On day 7 from the start of the cultivation, the mediumis removed by suction and the cells are fixed with an acetone:methanol(1:1) solution, to thereby allow tartaric acid-resistant acidphosphatase-positive cells stained. That is, 0.4 ml of a 0.2-M acetatebuffer (pH 5.2) containing a staining fluid (0.1 mg/ml Naphthol AS-MXphosphate (Sigma AB), 0.6 mg/ml Fast red violet LB sal (Sigma AB) and 20mM tartaric acid is added to each well and the resultants are allowed toreact at 37° C. for 1 hour. After color development, the number oftartaric acid-resistant acid phosphatase-positive cells, which is anindex of differentiation into osteoclasts, is measured under amicroscope.

When the test sample contains a substance that is effective forinhibition of osteoclast differentiation, the occurrence of tartaricacid-resistant acid phosphatase-positive cells is inhibited. Fromchanges in the above-mentioned number of cells depending on theconcentration of the test sample, an effective concentration range ofthe test sample can be obtained.

The substances F-1490 and F-1490-A of the present invention inhibitedthe differentiation of bone marrow cells into osteoclasts in lowconcentrations as shown in Table 1 below. TABLE 1 Inhibition rate ofdifferentiation Concentration into osteoclasts (%) (μg/ml) SubstanceF-1490 Substance F-1490-A 0 0 0 0.20 6 2 0.78 50 63 3.10 69 74 12.50 9887 50% growth inhibition 0.78 0.65 concentration (μg/ml)

Further, the substances F-1490 and F-1490-A of the present inventionshowed substantially no toxicity to various kinds of cultivated cells asshown in Table 2 below. TABLE 2 50% growth inhibition concentration(μg/ml) Cells Substance F-1490 Substance F-1490-A L1210 >50 >50Colon26 >50 >50 B16BL6 >50 >50

Note that the tests were performed as follows. Various kinds of murinecancer cells (L1210 (leukemia)), Colon 26 (colon cancer), B16BL6(malignant melanoma)) were suspended in RPMI1640 medium containing 10%fetal calf serum and adjusted to 2×10⁴ cells/well and 0.1 ml of each ofthe suspensions was seeded on a 96-well plate. At the same time, apredetermined amount of the substance F-1490 was added and theresultants were cultivated at 37° C. for 48 hours in the air containing5% CO₂. Further, 10 μl of 0.5% MTT (3-[4,5-dimethylthiazol-2-yl]2,5-biphenyl tetrazolium bromide) was added and the resultants werecultivated for 4 hours. After the cultivation, 0.1 ml of 10% SDS-0.01Nhydrochloric acid was added and absorbance at 570 nm was measured todetermine 50% growth inhibition concentration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing an infrared absorption spectrum of thesubstance F-1490 by the KBr method.

FIG. 2 is a diagram showing an ultraviolet absorption spectrum of thesubstance F-1490 measured in a methanol solution.

FIG. 3 is a diagram showing ¹H-nuclear magnetic resonance spectrum ofthe substance F-1490 measured in a heavy pyridine solution.

FIG. 4 is a diagram showing ¹³C-nuclear magnetic resonance spectrum ofthe substance F-1490 measured in a heavy pyridine solution.

FIG. 5 is a diagram showing ¹H-nuclear magnetic resonance spectrum ofthe substance F-1490-A measured in a heavy methanol solution.

FIG. 6 is a diagram showing ¹³C-nuclear magnetic resonance spectrum ofthe substance F-1490-A measured in a heavy methanol solution.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described by examples.However, the present invention should not be considered to be limited bythe following description.

EXAMPLE 1 Cultivation of the Substance F-1490-Producing Microorganismand Separation and Purification of the Substance F-1490

A loopful of a slant medium (potato dextrose agar) of Cunninghamella sp.F-1490 (FERM BP-8287) strain was inoculated in a 500-ml Erlenmeyer flaskcontaining 50 ml of a starter medium (2% potato starch, 1% glucose, 2%soybean powder (“Esusan Meat”, manufactured by Ajinomoto), 0.1%potassium dihydrogen phosphate and 0.05% magnesium sulfate, without pHadjustment) and incubated on a rotary shaker at 25° C. for 2 days toobtain a seed culture broth. A medium consisting of 4% glutinous starchsyrup, 2% peptone (manufactured by Kyokuto Pharmaceutical IndustrialCo., Ltd.), 1% yeastextract, 0.5% magnesium sulfate and 0.9% potassiumdihydrogen phosphate was used as a production medium. 60 ml each of themedium was charged in a 500-ml Erlenmeyer flask and sterilized. Then 1%each of the seed culture was inoculated thereto. The flasks wereincubated on a rotary shaker at 25° C. for 4 days and 20 liters of theobtained culture broth was centrifuged to separate it into a culturefiltrate and fungus body.

After adjusted to pH 2 with 6N hydrochloric acid, the obtained culturefiltrate was passed through a 5-liter column packed with adsorbentresin, Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation)equilibrated with water. The HP-20 column on which the active ingredientadsorbed was washed with 10 liters of 20% aqueous methanol solution andthen the active ingredient was eluted with 10 liters of methanol. Themethanol was evaporated from the eluted solutions by using an evaporatorand 2 liters of ethyl acetate was added to the residue and the resultantwas stirred. The ethyl acetate layer was taken up and 2 liters of waterwas added thereto. While stirring, the mixture was adjusted to pH 8 with1N aqueous sodium hydroxide solution. After leaving the mixture at restto allow it to be separated into two layers, the water layer wasseparated and adjusted to pH 2 with 1N hydrochloric acid. Further, after2 liters of ethyl acetate was added to effect extraction, the ethylacetate layer was concentrated under reduced pressure to obtain 6.1 g ofa brown oily substance.

The brown oily substance was dissolved in a small amount of methanol andthe solution was coated on silica gel. The resultant was packed in a400-ml silica gel column filled with chloroform. After it was washedwith 1 liter of a chloroform-methanol mixed solution (15:1), the columnwas eluted with 1 liter of a chloroform-methanol mixed solution (10:1).The thus obtained active fraction was concentrated under reducedpressure to obtain 0.39 g of a yellow oily substance. Further, this wasdissolved in a small amount of methanol and the solution was coated onsilica gel and charged in a silica gel column (50 ml) filled withtoluene. After it was washed with 150 ml of a toluene-acetone mixedsolution (3:1), the column was eluted with 150 ml of a toluene-acetonemixed solution (2:1). Fractions containing the active substance werecollected and concentrated under reduced pressure to obtain 106 mg of ayellow powdery substance.

The thus obtained yellow powdery substance was dissolved in a smallamount of methanol and charged in a 200-ml Sephadex LH-20 column(manufactured by Pharmacia AB) and eluted with methanol. The activefractions were collected and concentrated under reduced pressure toobtain 20 mg of the substance F-1490.

EXAMPLE 2 Synthesis of the Substance F-1490-A

To a suspension of 100 mg of 3-hydroxyanthranilic acid and 400 mg of1,2-cyclohexanedione in 20 ml of methanol was added 1 g of sodiumborohydride in a small amount at a time over 30 minutes underice-cooling with stirring. After completion of the addition, the mixturewas stirred at room temperature for 1 hour and then ice-cooled again.The resultant was adjusted to pH 7 by addition of 1N hydrochloric acidand the solvent was evaporated under reduced pressure. The obtainedresidue was purified by preparative TLC (hexane-ethyl acetate 1:1) toobtain a single band that develops color at an Rf value of 0.6 withphosphorus molybdenum sulfate under heating to obtain 90 mg of thetarget substance F-1490-A.

¹H-NMR and ¹³C-NMR spectra indicated that the substance F-1490-A has achemical structure shown below.

INDSUTRIAL APPLICABILITY

Since the substances F-1490 and F-1490-A have excellent osteoclastdifferentiation inhibiting activity and low toxicity to cultivatedcells, it is anticipated that they can be utilized as therapeutic agentsfor diseases that accompany an increase in the activity of osteoclasts.

1. A compound represented by formula (I):

wherein X represents —O— or —CH₂—, and R represents a hydroxyl groupwhen X represents —O—, or a hydrogen atom when X represents —CH₂—, orsalts thereof.
 2. The compound according to claim 1, wherein in formula(I), X represents —O— and R represents a hydroxyl group, or saltsthereof.
 3. The compound according to claim 1, wherein in formula (I), Xrepresents —CH₂— and R represents a hydrogen atom, or salts thereof. 4.A process for producing the compound according to claim 2, comprisingcultivating a microorganism that belongs to the genus Cunninghamella andis capable of producing the compound according to claim 2, andrecovering the compound according to claim 2 from the resultant culturebroth.
 5. Cunninghamella sp. F-1490 strain (FERM BP-8287) having acapability of producing the compound according to claim
 2. 6. Anosteoclast differentiation inhibitory agent comprising as an activeingredient the compound or salts thereof according to claim 1.